The long term aim of this proposal is to understand the molecular mechanisms underlying titin elasticity and its regulation. Titin is a giant modular protein that through alternative splicing of 363 exons, codes for a wide variety of isoforms that determine the elasticity of the hundreds of muscle types found in the human body. The predominant isoform of cardiac titin is a 3 MDa protein whose dynamic response to mechanical load is determined via two broad classes of modules in the elastic I band region: individually folded immunoglobulin like (Ig) domains and the elastomeric regions PEVK and N2B. We have pioneered polyprotein engineering and single molecule AFM studies of the mechanical properties of these modules, successfully generating a coarse grained view of the elasticity of titin. However, in our earlier experiments the force and length of single protein molecules varied simultaneously, preventing the development of a complete quantitative description of titin elasticity. To address this shortcoming we have developed the new force-clamp spectroscopy technique that monitors the length dynamics of a protein held under a constant force. Similar to the voltage-clamp techniques that revolutionized the study of ion channels, force-clamp now permits a deeper and more detailed study of force-dependent reactions in titin. For example, these new techniques now permit us to study the effect of mechanical forces on thiol/disulfide exchange, the common chemical reaction that regulates disulfide bonding in titin and in many other proteins. We will use these methods to study the force-dependent activity of the enzyme thioredoxin, a ubiquitous regulator of cellular redox states via disulfide bond reduction. These studies will not only uncover novel mechanisms of regulating titin elasticity but will also help develop a new structural biological technique that can probe enzyme dynamics during catalysis, with sub-Angstrom resolution, currently not possible with any other method. Force-clamp techniques also permit a detailed examination of the molecular forces driving the collapse of an extended protein, the most crucial component of titin elasticity whose origin remains unresolved. We will use force-clamp protocols combined with solvent substitution to study the recoil forces of the PEVK, N2B and of the unfolded Ig domains of titin. These experiments will test the hypothesis that the elasticity of unfolded titin modules is determined mainly by hydrophobic collapse of the extended polypeptides, challenging current paradigms of titin extensibility. Finally, through the use of force-clamp spectroscopy we will redefine the concept of mechanical stability to quantitatively compare titin Ig domains, first examining the mechanical properties of the titin N-terminus end domains Z1 and Z2. We will then test the hypothesis that the Z1Z2-telethonin complex forms a mechanically stable anchor between antiparallel titin molecules, and further probe the mechanical role of mutations in this complex linked to human disease. The proposed studies will examine the fundamental molecular mechanisms underlying titin elasticity, as well as develop a new form of protein spectroscopy to track protein dynamics in the Angstrom scale. PUBLIC HEALTH RELEVANCE Muscle elasticity is set by the giant protein titin. It is then of great medical importance to understand the mechanical properties of this protein. Studying protein mechanics has necessitated a novel set of tools at the nanoscale to study how single titin protein respond to a mechanical force. These studies will permit the development of mechanistic models of the elasticity of tissues such as heart muscle, in normal and diseased states.